Opteia il 6

Complementation of the THP-1 IRAK3^−/− knockout cell line (K6-3) with an empty pcDNA6.2/C-EmGFP-DEST plasmid (control) or with the plasmid encoding eGFP-tagged wild type IRAK3 or selected mutated versions of the protein (R97A, D377A, D385A, R97A/R372L) was performed as described previously [27 (link)]. Transiently transfected IRAK3 knockout cells were treated with 1 µg mL⁻¹ LPS with or without 0.1 nM 8-Br-cGMP for 24 h. Levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured using BD OptEIA IL-6 and OptEIA TNF-α ELISA kits (BD Biosciences) according to the manufacturer’s instructions. Data from 7–9 independent experiments were normalized to the eGFP fluorescence signal (excitation: 470–15 nm, emission: 515–20 nm) measured by CLARIOstar Plus and total protein concentration quantified by the Bradford method [58 (link)] and analyzed (p value < 0.05; one-way ANOVA followed by Turkey’s multiple comparisons test).

Wild type and IRAK3 knockout/knockdown THP-1 cells were split at approximately 2 × 10⁴ cells in 300 µL media per well in Corning^® Costar cell culture clear flat bottom 96 well plates (Sigma-Aldrich^®, Macquarie Park, Australia). Cells were treated with 1 × PBS as the negative control, 1 µg/mL lipopolysaccharide (LPS, Escherichia coli 055:B5; Merck, Bayswater, Australia) as the positive control, or 3.0 × 10⁶ plaque forming units (PFU) per mL of crudely purified or highly purified (see above) suspensions of bacteriophages KPN7 or KPN8 for 24 h. 100 µL of cell supernatants were then collected and measured for concentrations of human cytokines (TNF-α and IL-6) using BD OptEIA™IL-6 and OptEIA™TNF-α ELISA kits (BD Biosciences, North Ryde, Australia) according to the manufacturer’s instructions.

Wild type and IRAK3 knockdown THP-1 cells were cultured at approximately 1 × 10⁵ to 2 × 10⁵ cells in 500 μL medium per well in Corning Costar cell culture clear flat bottom 24-well plates. After cell treatments with 1 µg/mL LPS and the addition of 100 fM–100 nM 8-Br-cGMP or 0.1 nM–10 µM ODQ or 0.1–100 µM DEA NONOate (control cells treated with LPS only), 100 µL of supernatant aliquots from cell cultures were collected and measured for the level of human cytokines (TNF-α and IL-6) using BD OptEIA IL-6 and OptEIA TNF-α ELISA kits (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions.